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Originally published In Press as doi:10.1074/jbc.M412593200 on November 24, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7962-7975, March 4, 2005
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DRO1, a Gene Down-regulated by Oncogenes, Mediates Growth Inhibition in Colon and Pancreatic Cancer Cells*

Guido T. Bommer{ddagger}, Claudia Jäger{ddagger}, Eva-Maria Dürr{ddagger}, Sebastian Baehs{ddagger}, Sören T. Eichhorst{ddagger}, Thomas Brabletz§, Gang Hu¶||, Thomas Fröhlich**, Georg Arnold**, Dagmar C. Kress{ddagger}{ddagger}, Burkhard Göke{ddagger}, Eric R. Fearon¶, and Frank T. Kolligs{ddagger}§§

From the {ddagger}Department of Medicine II, University of Munich, 81377 Munich, Germany, the §Institute of Pathology, University of Erlangen, 91054 Erlangen, Erlangen, Germany, the Division of Molecular Medicine & Genetics and the Cancer Center, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, the **Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, University of Munich, 81377 Munich, Germany, and the {ddagger}{ddagger}Institute of Molecular Animal Breeding and Biotechnology, University of Munich, 81377 Munich, Germany

Received for publication, November 8, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neoplastic progression in human tissues appears to be paralleled by a series of genetic and epigenetic alterations. In human colorectal cancers, defect Wnt/{beta}-catenin/T-cell factor and RAS/RAF signaling pathways have a major contributing role in tumor initiation and progression. To date, much of the research on the consequences of {beta}-catenin activation has been focused on genes whose expression is believed to be activated by {beta}-catenin-associated T-cell factor-dependent transcription. Little is known about genes whose expression may be down-regulated secondary to {beta}-catenin activation. Using a subtractive suppression hybridization approach, we identified a gene with markedly decreased expression in rat RK3E epithelial cells neoplastically transformed by {beta}-catenin. Because expression of this gene was also down-regulated in RK3E transformed by several other oncogenes, the gene was named DRO1 for "down-regulated by oncogenes 1." Compared with corresponding normal tissues, DRO1 expression was found to be very reduced in colon and pancreatic cancer cell lines as well as in most colorectal cancer specimens. The predicted DRO1 protein contains three repetitive elements with significant similarity to the carboxyl-terminal regions of the predicted proteins from DRS/SRPX/ETX1 and SRPUL genes, suggesting the existence of a new protein family. Ectopic expression of DRO1 in neoplastically transformed RK3E or colorectal and pancreatic cancer cell lines lacking endogenous DRO1 expression resulted in substantial inhibition of growth properties. DRO1 was found to suppress anchorage independent growth and to sensitize cells to anoikis and CD95-induced apoptosis. Our findings suggest that inhibition of DRO1 expression may be an important event in the development of colorectal and pancreatic cancers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In aggregate, gastrointestinal cancers constitute a major cause of tumor-related morbidity and mortality. A series of genetic and epigenetic events accompanies the conversion of a normal epithelial cell to a malignant cancer cell (1–3). Analysis of the sequential occurrence of these aberrations in colorectal cancer development led to a working genetic model of a stepwise carcinogenesis in epithelial tissues (4). Defects in the Wnt/APC/{beta}-catenin/TCF signaling pathway are believed to play a leading role in the initiation of colorectal carcinogenesis (reviewed in Refs. 5 and 6). In up to 80% of colorectal cancers inactivating mutations in the adenomatous polyposis coli (APC)1 tumor suppressor gene can be seen (7–9). In a subset of the cancers with intact APC function, gain-of-function mutations affecting the amino-terminal region of {beta}-catenin are found (10, 11). Loss-of-function defects in APC or gain-of-function mutations in {beta}-catenin lead to the stabilization of the {beta}-catenin protein, which consequently accumulates in the cytoplasm and the nucleus where it can bind to and activate the transcriptional activity of members of the T-cell factor (TCF) family of transcription factors. Besides colorectal cancer, defects in {beta}-catenin regulation are found in other tumors, such as hepatoblastoma (12, 13), hepatocellular carcinoma (14, 15), and intestinal-type gastric cancer (16).

Only a small subset of pancreatic adenocarcinomas harbor similar defects in the Wnt/APC/{beta}-catenin/TCF signaling pathway, i.e. the acinar-cell carcinoma and solid pseudopapillary tumors of the pancreas (17, 18). Genetic alterations of the most common histological type of pancreatic cancer, i.e. ductal adenocarcinoma, more often involve multiple changes in the protooncogene K-Ras as well as the tumor suppressor genes CDKN2A, p53, and SMAD4/DPC4 (reviewed in Ref. 3).

A sizeable number of genes whose expression is increased in colorectal cancer cells with constitutively activated {beta}-catenin/TCF function have been described (reviewed in Ref. 6), including well known protooncogenes like c-MYC (19), cyclin D1 (20, 21), and candidate protooncogenes like ITF2 (22), as well as negative regulators like connexin 43 (23) and Axin-2/conductin (24–26). Much less data have emerged on the role of genes down-regulated during transformation by different oncogenes. So far, only three genes down-regulated secondary to activated {beta}-catenin/TCF signaling have been identified: DRCTNNB1A (27), MCP-3 (28), and IGFBP-6 (29). Whereas evidence for a direct influence of {beta}-catenin/TCF on the expression of MCP-3 and IGFBP-6 has been offered, insights into how {beta}-catenin/TCF might mediate effects on DRCTNNB1A expression are lacking.

Transformation of immortalized cell lines is often used as a model system for carcinogenesis. Candidate downstream targets of oncogenes have been identified by comparing the transcriptional status of parental cell lines with cell lines transformed by different oncogenes. To achieve a more comprehensive understanding of the effects of deregulated {beta}-catenin/TCF signaling in cancer, we used a subtractive suppression hybridization approach to identify transcripts whose expression was down-regulated following {beta}-catenin-mediated neoplastic transformation of RK3E cells, an E1A-immortalized rat kidney epithelial cell line (30). Our efforts led to the identification of a gene with markedly decreased expression not only in RK3E cell lines transformed by {beta}-catenin and several other oncogenes, but also in gastrointestinal cancer cell lines and primary tumor specimen. We describe here the analysis of this gene termed DRO1 (down-regulated by oncogenes 1) in normal and cancer tissues as well as studies of the functional consequences of restoring DRO1 gene expression in colon and pancreatic cancer cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of DRO1—Subtractive suppression hybridization was performed using a PCR-based technique (31) (PCR-Select, Clontech, Palo Alto, CA). mRNA from the {beta}-catenin-transformed cell line RK3E/{beta}-catenin-S33Y-A (32) was used as driver, mRNA of parental RK3E (30) cells served as tester. One clone was isolated that was consistently down-regulated in multiple independent {beta}-catenin-transformed RK3E clones. Full-length rat and human cDNA sequences were obtained by screening a rat brain cDNA library (Stratagene, La Jolla, CA) and a human heart cDNA library (Stratagene), using standard techniques. The rat and human sequences of DRO1 were deposited in GenBankTM (accession numbers AY548105 [GenBank] , AY548106 [GenBank] , and AY548107 [GenBank] ).

The entire open reading frame of human DRO1 was amplified with PfuI polymerase (Stratagene) using the oligonucleotide primers: DRO1 forward, 5'-TGAATTCCACCATGACATGGAGAATGGGACC-3' and DRO1 reverse, 5'-ACTCGAGTCAGCTAGCGTAAGGGTATCCATGGTGATA-3' with the human cDNA clone as template DNA. The PCR product was subcloned into the EcoRI and XhoI sites of the cytomegalovirus-promoter driven expression plasmid pCDNA3 (Invitrogen). An artificial carboxyl-terminal hemagglutinin (HA) tag was introduced by ligating the annealed oligonucleotides HA forward, 5'-CTAGCGGCTACCCCTATGATGTGCCAGACTACGCCTGAC-3' and HA reverse, 5'-TCGAGTCAGGCGTAGTCTGGCACATCATAGGGGTAGCCG-3' into the corresponding NheI and XhoI restriction sites. The sequence of the resulting plasmid, pCDNA3-DRO1-HA, was confirmed by automated sequencing. For retroviral transduction the cDNA was digested with BamHI and XhoI and ligated into the BamHI and SalI sites of the plasmid pBABE-puro 33. pC{beta}A-bGH contains a genomic BamHI-BglII fragment of the bovine growth hormone (bGH) gene driven by the chicken {beta}-actin promoter (supplied by E. Wolf, Institute of Molecular Animal Breeding and Biotechnology, University of Munich, Munich, Germany).

The putative promoter region of DRO1 was amplified by PCR from the human BAC-clone RP11-36I23 (library RPCI-11 from Roswell Park Cancer Institute created by M. Tateno and K. Osoegawa; obtained from RZPD, Berlin, Germany) by using the primers 5'-ACTCTAGAGCTGGGGATCCTGAACGATACACT-3' and 5'-TCGAGTCAGGCGTAGTCTGGCACATCATAGGGGTAGCCG-3' introducing sites for XbaI and XhoI. The obtained PCR product was subcloned into the NheI and XhoI sites of pGL3-basic (Promega, Mannheim, Germany) resulting in the construct 4235. By deleting sequences upstream of an EcoRV restriction site, an AvrII site, and a KpnI site, constructs 3607, 3135, and 1166 were generated, respectively. Synthetic oligonucleotides were obtained from MWG-Biotech, Ebersberg, Germany.

Cell Culture—Human colorectal cancer cell lines SW948, SW620, Caco2, LS513, HT29, RKO, HCT116, SW48, WiDR, DLD1, SW480, SW403, and the cell line Colo320 (generous gifts of H. Lahm, University of Heidelberg, Heidelberg, Germany, and G. van Kaick, DKFZ, Heidelberg) as well as COS, 293T, RK3E, and the RK3E-derived lines, the generation of which has been described previously (32, 34), were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum. Human pancreatic cancer cell lines CFPAC-I, Panc I, Hs766T, MIAPaCa-2, and BxPc3 were obtained from the American Type Culture Collection (Manassas, VA) and cultured as recommended. The plasmid pBABEpuro-DRO1-HA and control plasmid pBABEpuro were used to generate amphotropic viruses as described previously (32, 33). Stable monoclonal cell lines were obtained by limiting dilution of cells 48 h after transfection with pCDNA3-DRO1-HA or control vector pCDNA3 in the presence of G418 (Invitrogen) or 24 h after infection with virus supernatant in the presence of puromycin (Sigma). For cloning efficiency assays HCT116, SW480, PancI, and BxPc3 cells were either transfected with equal amounts of pCDNA3-DRO1-HA or the corresponding empty vector. 48 h post-transfection of the plasmids 5 x 105 cells were plated on 10-cm dishes and cultured with medium supplemented with G418 (Invitrogen) for selection of transfected cells. Colonies were stained (1.5% glutaraldehyde and 0.06% methylene blue in Hanks' balanced salt solution (Invitrogen)) and photographed. Assays of colony formation in soft agar were performed essentially as described previously (32). For analysis of N-glycosylation of DRO1, COS cells were transfected with pCDNA3-DRO1-HA or empty pCDNA3 12 h prior to treatment with 1 µg/ml tunicamycin (Sigma) for 12 h. Similarly, for analysis of secretion of DRO1, cells were transfected with pCDNA3-DRO1-HA, pC{beta}A-BGH, and pCDNA3. Serum-free medium was added 12 h after transfection. Supernatant was harvested 36 h later and centrifuged to remove cellular debris. To assess the influence of methylation on the DRO1 promoter, cells were treated for 96 h with 2 µM 5-azacytidine (Sigma) with daily replacement of growth medium. For anoikis assays cells were trypsinized into single cell suspensions. To maintain cultures in suspension 105 cells were given in 100-mm non-tissue culture plates to which cells could not attach.

Reporter Assays—Colorectal cancer cell lines were transfected with 0.4 µg of Firefly reporter plasmid and 0.1 µg of Renilla luciferase control plasmid (pRL-TK, Promega). RK3E cells and RK3E-derived transformed cell lines were transfected with 0.25 µg of luciferase reporter plasmid and as control for transfection efficiency with 0.25 µg of constitutively active {beta}-galactosidase reporter plasmid pCH110 (Amersham Biosciences). Firefly and Renilla luciferase were measured with Promega's Dual-Glo luciferase kit according to the manufacturer's recommendations by using a luminescence reader (Turner, Sunnyvale, CA). {beta}-Galactosidase activity was measured as described previously (32).

RNA Preparation and Northern Blotting—Tissue culture cells were harvested in TRIzol (Invitrogen) for RNA extraction. Organs from a 4-week-old Wistar rat were placed in TRIzol and immediately homogenized with a standard tissue homogenizer (Miccra, Muehlheim, Germany). Normal pancreatic tissue samples as well as matched pairs of normal colon and colorectal adenocarcinoma were microdissected using an adjacent hematoxylin and eosin-stained section as guidance from frozen primary tissue samples. For Northern blots total RNA (10 µg) was separated on 1.2% formaldehyde-agarose gels and transferred to Hybond N+ membranes (Amersham Biosciences) by capillary action. A 1124-bp HindIII fragment of rat DRO1 cDNA and a 425-bp PCR product of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (forward primer 5'-ACCACAGTCCATGCCATCAC-3'; reverse primer 5'-TCCACCACCCTGTTGCTGTA-3') were labeled with [32P]dCTP by random priming. Northern blot hybridization to 32P-labeled probes was carried out by standard methods. Signals were detected by exposure to Bio-Max film (Amersham Biosciences) at -80 °C with an intensifying screen.

PCR—Following DNase treatment, RNA was reverse transcribed with random hexamer primers (final concentration 2.5 ng/µl) using SuperScriptTM II reverse transcriptase as described by the manufacturer (SuperScriptTM First-Strand Synthesis System for RT-PCR, Invitrogen). Quantitative PCR was performed in a Rotorgene RG-3000 (Corbett Research, Sidney, Australia) real-time PCR system using SYBR-Green I dye (Molecular Probes) for detection. Primers for human and rat DRO1 were hDRO1 forward, 5'-CCTGGGCAGCGAGAAGAAGAAAG-3'; hDRO1 reverse, 5'-CCGGGATGGAGGGTAAAGATT-3'; and rDRO1 forward, 5'-ACAGGCCTGGGCAGTGACAAG-3'; rDRO1 reverse, 5'-CCGGAGGCCTATGGGAGAC-3', respectively. For normalization of PCR, the human APP gene, the human GAPDH gene, and the rat Gapdh gene were used. APP primers have been previously published (35). For human and rat cDNAs the same GAPDH primers were used: GAPDH forward, 5'-ACCACAGTCCATGCCATCAC-3' and GAPDH reverse, 5'-TCCACCACCCTGTTGCTGTA-3'. PCR was performed using 1.25 units of Platinum TaqDNA Polymerase (Invitrogen), a final concentration of 2.5 mM MgCl2, and SYBR-green I diluted to 1:50,000. PCR efficiency was determined by analyzing serial dilutions of cDNA. Relative expression levels were calculated with QGene software (36). Pair-wise fixed reallocation randomization test was used to determine the significance of differences between samples (37).

Western Blotting—Cells were lysed in RIPA lysis buffer (Tris-buffered saline, 0.5% deoxycholate, 0.1% SDS, 1% Nonidet P-40) supplemented with proteinase inhibitors (Complete Proteinase Inhibitors, Roche). The protein concentrations were determined by a modified Bradford assay (Bio-Rad Protein Assay). Equal amounts of protein were separated by electrophoresis in discontinuous SDS-polyacrylamide gels. After transfer of the proteins to Immobilon P membranes (Millipore, Schwalbach, Germany), and blocking in 10% nonfat dry milk (Bio-Rad) the antibody incubation steps were performed in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk. High affinity monoclonal anti-HA tag antibody (clone 3F10, Roche), anti-plakoglobin, and anti-{beta}-catenin antibodies (Transduction Laboratories, Lexington, KY) and anti-caspase 3 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) were used at 1:1,000 dilutions, respectively. Anti-caspase 8 antibody (a generous gift of P. Krammer, DKFZ, Heidelberg, Germany) and the anti-actin antibody (clone AC-15, Sigma) were used at 1:100 and 1:10,000 dilutions, respectively. The chicken IgY antibody against bGH was a generous gift of M. Erhard, Institute of Animal Welfare, Ethology and Animal Hygiene, University of Munich, Munich, Germany.

The DRO1-peptide IERSTLEEPNLQPLQRR (corresponding to aa 61 to 77 of human DRO1) was synthesized and coupled to a peptide carrier to increase immunogenicity. Purification was performed by high performance liquid chromatography and accuracy of peptide composition was monitored by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (provided by F. Lottspeich, MPI-Biochemie, Munich, Germany). Rabbits were immunized with the peptide according to standard protocols. Serum was affinity purified by using NHS-activated Sepharose (Amersham Biosciences) coupled to the peptide that had been used for immunization.

The secondary horseradish peroxidase-conjugated antibodies goat anti-rat (Santa Cruz Biotechnology), goat anti-mouse (Amersham Biosciences), goat anti-rabbit (Amersham Biosciences), and rabbit anti-chicken IgG (Sigma) were used at dilutions of 1:10,000. The blots were subjected to enhanced chemiluminescence (ECL, Amersham Biosciences) and exposed to Hyperfilm ECL (Amersham Biosciences).

Immunofluorescence—COS cells were grown on acid-washed coverslips. 48 h after cotransfections of pCDNA3-DRO1-HA and expression plasmids for markers of the endoplasmic reticulum or the Golgi stack (SEC61-YFP and Gal-N-Ac-T2-YFP (38)), coverslips were washed with PBS, fixed with 3% paraformaldehyde (Sigma) in PBS for 15 min, and then permeabilized for 15 min in 0.5% Triton X-100 (Sigma) and 1% goat serum (Invitrogen) in PBS. Prior to incubation with the primary antibody, coverslips were blocked for 30 min by treatment with 20% goat serum, 0.2% Triton X-100 in PBS. After washing with 2% goat serum, 0.2% Triton X-100 in PBS, the coverslips were incubated for 2 h with the anti-HA tag antibody in 2% goat serum, 0.2% Triton X-100 in PBS. After further washes, the coverslips were incubated with a secondary goat anti-rat IgG antibody conjugated with Cy3 (Dianova, Hamburg, Germany) at 1:500 and for nuclear staining with Hoechst 33342 (Sigma) at a final concentration of 20 µg/ml for 30 min. The coverslips were washed with PBS and mounted on slides using Vectashield mounting medium (Vector Laboratories, Bulingame, CA). Confocal fluorescence microscopy was performed on a Zeiss LSM2100 Meta laser scanning microscope (Carl Zeiss, Jena, Germany) using optical sections of 2 µm.

Apoptosis Assays—Cells were harvested 24 h after the start of suspension cultures or 24 h after start of treatment with agonistic APO-1/CD95 antibody (a generous gift of P. Krammer, DKFZ, Heidelberg, Germany) at increasing concentrations. To optimize receptor cross-linking in these experiments, protein A (Sigma) was added at 1/100 the concentration of the antibody. For quantification of DNA fragmentation and cell cycle distribution, cells were scraped in PBS, centrifuged at 800 x g and lysed in hypotonic lysis buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 µg/ml propidium iodide (39). After overnight incubation at 4 °C nuclei were analyzed for DNA content by flow cytometry using FACSCALIBUR and CellQuest software (BD Biosciences). For caspase 3 activity assays cells were harvested and lysed after 24 h in suspension. Lysates were stored at -80 °C until the assay was performed. An aliquot of the precleared supernatant was mixed with the caspase-3 Z-DEVD-AFC-substrate (Calbiochem, Schwalbach, Germany) and incubated at 37 °C. Fluorescence was measured by a fluorometer at an excitation wavelength of 450 ± 50 nm and a detection wavelength of 530 ± 25 nm. The level of caspase-3 activity was normalized to the amount of protein in each sample.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of DRO1 as a Gene That Is Down-regulated by Multiple Oncogenes—A PCR-based suppression subtractive hybridization approach (31) was performed to identify genes whose expression was markedly decreased in RK3E epithelial cells neoplastically transformed by expression of a mutant stabilized form of {beta}-catenin (S33Y-{beta}-catenin). Based on an initial analysis of the expression patterns of 6 clones from a library of potentially down-regulated genes, we focused attention on a 675-bp cDNA fragment that showed markedly decreased transcript levels in 10 of 10 {beta}-catenin-transformed RK3E cell lines and 9 of 9 {gamma}-catenin-transformed RK3E lines (34) (Fig. 1, A and B, and data not shown). To test whether down-regulation of this gene was specific for {beta}- and {gamma}-catenin-induced transformation or represented a general consequence of oncogenic transformation of the RK3E cells, we assessed expression of the identified gene in RK3E cells transformed by other oncogenes including K-Ras, H-Ras, c-MYC, and GLI (Fig. 1, A and B). Because our Northern blot and quantitative RT-PCR analyses revealed down-regulation of the gene in all neoplastically transformed RK3E lines, regardless of the oncogene used to induce transformation, the gene was termed DRO1. DRO1 expression was detected ubiquitously in all rat organs tested (Fig. 1C). To analyze expression of DRO1 in human tissues RT-PCR studies were performed. High DRO1 expression was found in both colonic mucosa and whole pancreas preparations (Fig. 1D, lanes 1 and 3) as well as in microdissected colonic epithelium and pancreatic ductal epithelium (Fig. 1D, lanes 2 and 4). This demonstrates that DRO1 is expressed in epithelial tissues that are frequently affected by neoplastic transformation associated with deregulation of Wnt and Ras/Raf signal transduction.



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  		FIG. 1.
DRO1 expression is suppressed in transformed RK3E cell lines. A, Northern blot studies of DRO1 expression were carried out on total RNA from parental RK3E cells and oncogene-transformed RK3E lines. After hybridization with the DRO1 probe, the membrane was stripped and rehybridized with a GAPDH probe to control for loading of the lanes. B, DRO1 expression levels in the transformed RK3E lines were determined by real-time PCR and compared with DRO1 expression in parental RK3E cells. Expression levels were normalized to GAPDH expression levels. Values shown represent the mean ± S.E. of duplicate experiments. C, expression of DRO1 in normal rat tissues was assessed by quantitative PCR. Expression levels were normalized to GAPDH expression levels. Values shown represent the mean ± S.E. of duplicate experiments. D, DRO1 expression in human colon and pancreas was analyzed by RT-PCR. Colonic epithelium and pancreatic ductal epithelium express DRO1 at high levels. GAPDH expression is shown as a control.

 
Rat brain and human heart cDNA libraries were screened to isolate full-length clones of the rat and human transcripts of DRO1. cDNAs for two human transcripts of 3822 and 3988 base pairs, respectively, were isolated, both of which encoded an open reading frame of 950 amino acids. The single isolated rat transcript comprised 3604 base pairs with an open reading frame of 949 amino acids. The predicted rat and human DRO1 proteins showed 86.3% amino acid identity. Comparison of the identified transcripts with published genome sequences (NCBI Blast) revealed similar gene structures in rat and man. In both species, the DRO1 gene comprises 8 exons. The human DRO1 gene is located on chromosome 3q13.2 and the rat gene is located on chromosome 11q21. Data bank analyses revealed high homology of the rat and human DRO1 sequences to the mouse gene Urb (40) and the chick gene Equarin-L (41), suggesting rat and human DRO1 might be the orthologues of these genes.

DRO1 Is a Post-translationally Modified Protein That Shows Homology to the Putative Tumor Suppressor DRS—DRO1 amino acid sequence analysis revealed three internal repeats (IR1-IR3) of 142 to 147 amino acids (human IR1 aa 140–281, human IR2 aa 614–760, human IR3 aa 770–913; rat IR1 aa 140–281, rat IR2 aa 613–759, rat IR3 aa 769–912). The three repeats represent the most highly conserved regions of the predicted DRO1 proteins when the human, rat, and mouse sequences are compared (identical amino acids between 93 and 97%; Fig. 2A). In addition, the repeats show almost 30% amino acid sequence identity to the carboxyl-terminal regions of the genes DRS/SRPX/ETX1 (42–44) and SRPUL (45) (Fig. 2, A and B). The DRS/SRPX/ETX1 (down-regulated by src/Sushi-repeat Protein on the X-chromosome) gene has been described as a gene that is down-regulated as a result of neoplastic transformation of fibroblastic cells by various oncogenes, including c-src (44). We found that Drs expression was also strongly reduced after neoplastic transformation of RK3E cells by {beta}-catenin, {gamma}-catenin, K-RAS, H-RAS, c-MYC, and GLI, ranging from 15 to 50% of the levels seen in parental RK3E cells (data not shown).



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  		FIG. 2.
DRO1 has three repetitive elements which show homology to Drs. A, schematic representation of the domain structure of human DRO1 in comparison to the human putative tumor suppressor gene DRS/SRPX/ETX1. Percentages represent the proportion of identical amino acids between internal repeats (IR) of DRO1 and the COOH-terminal region of Drs. B, DRO1 contains three internal repeats showing a significant homology to the carboxyl terminus of the putative tumor suppressor Drs/SRPX/ETX1 and to SRPUL. Identical amino acids of internal repeats (IR1 to IR3) between sequences of rat and man DRO1, mouse Urb, chicken Equarin L, and putative zebrafish DRO1 are shaded in black. Identity in more than 80% of sequences is shaded in gray.

 
After transient transfection of COS cells with an expression vector for DRO1 containing a carboxyl-terminal HA epitope tag (DRO1-HA), a single protein band was detected by Western blotting. In contrast to the predicted size of 109.5 kDa (i.e. 108 kDa for DRO1 and 1.5 kDa for the hemagglutinin tag), the expressed HA-immunoreactive protein migrated at a significantly larger size of 130 kDa (Fig. 3A). Sequence analysis (48, 49)2 predicted the presence of a signal peptide (amino acids 1–23 in man and amino acids 1–27 in rat) as well as several potential N-glycosylation sites (amino acids 667 and 835 in man; amino acids 467, 666, and 834 in rat). Additionally several potential mucin-type O-glycosylation sites were predicted in a threonin-rich region between amino acids 330 and 450. Consistent with its postulated N-glycosylation, treatment of cells with tunicamycin resulted in a significant decrease of the apparent molecular weight of the DRO1 protein in SDS-PAGE (Fig. 3A).



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  		FIG. 3.
DRO1 is post-translationally modified. A, ECL Western blot analysis with an anti-HA antibody was carried out on whole cell lysates of COS cells transiently transfected with pCDNA3-DRO1-HA (lanes 1 and 2) or empty vector (lane 3). 12 h after transfection cells were either treated with tunicamycin (lanes 2 and 4) or mock treated. To demonstrate equal loading of the lanes, the blot was stripped and ECL Western blotting with an anti-plakoglobin antibody was performed. B, Western blot analysis with an anti-HA (lanes 1–4, first row) and anti-bovine growth hormone antibody (bGH, lanes 5–8) was performed on whole cell lysates (lanes 1, 2, 5, and 6) and supernatants (lanes 3, 4, 7, and 8) of cells transiently transfected with pCDNA3-DRO1-HA (lanes 1 and 3), C{beta}A-bGH (lanes 5 and 7), or pCDNA3 (lanes 2, 4, 6, and 8). Membranes were stripped and reprobed with affinity purified antiserum against human DRO1 and {beta}-actin as indicated. C, COS cells were transiently transfected with an expression plasmid for DRO1-HA. DRO1-HA was stained with a Cy3-labeled secondary antibody (red) and nuclei were stained with Hoechst 33342 (blue). Confocal pictures were obtained showing DRO1 alone. The upper part of the picture is shown at a magnification of x630, the lower part is a magnification of the indicated box.

 

Because sequence analysis predicted a signal peptide, we next tested whether DRO1 might be a secreted protein. However, only minute amounts of the transfected DRO1 protein were detectable in the supernatant of COS cells transiently transfected with DRO1-HA or control vectors (Fig. 3B, first row, lanes 1–4). Similar results were obtained by using an affinity purified polyclonal antiserum against DRO1 (Fig. 3B, second row, lanes 1–4). In particular, no additional smaller bands were observed in the supernatant with the anti-DRO1 antiserum, indicating that the lack of detectable DRO1-HA in the supernatant is not because of post-translational cleavage of the HA tag. Identical results were obtained in 293T cells showing virtually no detectable DRO1 protein even after further concentration of the supernatant (data not shown). Detection of transiently transfected bovine growth hormone in the same cells suggests that the cells do not have a generalized blockade of the secretory pathway and secreted proteins can be readily detected in the supernatant (Fig. 3B, lanes 5–8). Immunofluorescence studies to analyze the subcellular distribution of DRO1 revealed a granular cytosolic pattern of staining (Fig. 3C). No DRO1 immunofluorescence was detected in the nucleus, nor was membranous DRO1 staining detected. Cotransfections of DRO1 with vectors expressing Golgi stack (Gal-N-Ac-T2-YFP (38)) and endoplasmic reticulum (Sec 61-YFP (38)) proteins showed only partial overlap with DRO1 expression sites (data not shown).

DRO1 Is Down-regulated in Human Cancer—RT-PCR studies revealed DRO1 expression in microdissected colonic and ductal pancreatic epithelium (Fig. 1D). We were therefore prompted to study DRO1 expression in human and pancreatic cancer cell lines by quantitative RT-PCR. Relative DRO1 mRNA expression was assessed in 12 colon cancer cell lines and six independent samples from microdissected colonic epithelium. Of the 12 colon cancer cell lines studied, only Caco2 cells revealed DRO1 expression greater than 5% of the levels seen in normal colon (Fig. 4A). The expression of DRO1 in five pancreatic cancer cell lines was compared with five independent samples from microdissected normal human pancreas. When compared with its expression in normal pancreas, DRO1 expression was substantially reduced or absent in all pancreatic cancer cell lines analyzed (Fig. 4B). Endogenous DRO1 protein could be detected by the DRO1 antiserum in extracts from normal colon but not in extracts from the cell lines analyzed (Fig. 4C and data not shown).



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  		FIG. 4.
DRO1 expression is down-regulated in human cancer cell lines and primary tumor tissue. DRO1 expression was determined by quantitative PCR and Western blot analysis in colorectal and pancreatic cancer cell lines as well as in primary tissues. A, relative DRO1 expression in colorectal cancer cell lines was determined by quantitative RT-PCR by comparison to six independent samples from colonic epithelium after normalization to APP expression. B, relative DRO1 expression in pancreatic cancer cell lines was determined by comparison to four independent pancreatic samples after normalization to GAPDH expression. Values represent percentage of control tissue expression levels. Error bars of cell lines represent standard deviations of duplicate measurements, whereas error bars of the independently measured normal tissues represent the standard error. C, protein extracts of colorectal and pancreatic cancer cell lines and normal colon were separated by gel electrophoresis and subjected to Western blot analysis. An identical blot was hybridized with antibodies against {beta}-catenin and {beta}-actin as control for equal loading of the lanes. D, cDNA from matched pairs of colorectal carcinoma and adjacent normal colorectal epithelium from 10 patients was subjected to quantitative real-time PCR with primers specific for DRO1. Expression levels were normalized to the expression levels of APP and are represented relative to the expression level of the corresponding normal tissue. Error bars represent standard deviations of triplicate measurements. Asterisks signify statistical significance p < 0.05 in pairwise fixed reallocation randomization test.

 
To test whether DRO1 expression is also down-regulated in primary human cancers, matched pairs of colorectal cancer samples and surrounding normal colonic epithelium from 10 individual patients were analyzed by quantitative PCR. For normalization APP expression levels were assessed, because APP expression levels have been shown not to differ between colorectal cancer and adjacent normal colon (35). DRO1 expression was significantly lower in six of the 10 cancer samples than in the matched normal tissues (Fig. 4D).

DRO1 Down-regulation Is Mediated by Transcriptional Repression—DNA methylation as a potential mechanism for down-regulation of DRO1 in cancer cells was studied. No regions fulfilling high stringency criteria for CpG islands were found in the putative DRO1 promoter (50). One CpG-rich region fulfilling low stringency criteria for CpG islands was identified slightly downstream of the presumptive translational start site in exon 2. High levels of DNA methylation of this region were found in all colorectal cancer cell lines analyzed, but also in DNA isolated from normal colonic epithelium (data not shown). Treatment of colon cancer cell lines lacking substantial DRO1 expression with the demethylating agent 5-azacytidine resulted in some increase in DRO1 expression (Fig. 5A). However, this expression still only corresponds to a fraction of the expression observed in normal colonic epithelium. As no relevant CpG islands could be identified within proximal promoter and upstream flanking sequences, the increase of DRO1 expression seen following 5-azacytidine treatment is more likely to be mediated by indirect, rather than direct effects of methylation of DRO1 promoter sequences.



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  		FIG. 5.
Analysis of DRO1 transcriptional regulation. A, colon cancer cell lines were treated with the demethylating agent 5-azacytidine and DRO1 expression was assessed by quantitative PCR normalized to the expression of APP (open bars). Filled bars represent DRO1 expression levels of mock treated cells. Mean values ± S.E. of a triplicate experiment are shown. B, schematic representation of DRO1 reporter constructs. Black boxes represent exon 1 and luciferase open reading frame. White boxes represent intron 1 and sequences upstream of the putative transcriptional start site (TSS). Numbers relate to the nucleotide position relative to the putative transcriptional start site. C, parental RK3E and transformed derivatives were transfected with the 4235 reporter construct or the control vector pGL3-basic. Luciferase activities were normalized for transfection efficiencies by measuring the activity of a cotransfected expression plasmid for {beta}-galactosidase. Values shown represent -fold activation of the 4235 reporter construct versus pGL3-basic control vector. The mean ± S.E. from three experiments are shown. D, colorectal cancer cell lines were transfected with reporter constructs 4235 and 1166 relative as well as empty pGL3 vector. Firefly luciferase activity was normalized to the activity of the cotransfected constitutively active expression vector for Renilla luciferase. Activity of the 4235 reporter construct was arbitrarily set to 1. Asterisks indicate highly significant differences of Colo320 to Caco2 (p = 0.0004), SW480 (p = 0.0001) and HCT116 (p = 0.0271) according to Student's t test. Error bars represent mean ± S.E. of 3 to 5 independent experiments. E, SW480 cells (left panel) and HCT116 cells (right panel) were transfected with reporter plasmids starting at the indicated positions upstream of the transcriptional start site. Luciferase activities were normalized to the activity of a cotransfected expression plasmid for Renilla luciferase and are represented here relative to the activity of the 4235 reporter construct. The mean ± S.E. from three experiments are shown.

 
To further analyze the potential mechanism of regulation of DRO1 expression by trans-acting factors, a luciferase reporter gene construct containing the regulatory region upstream of the presumptive DRO1 translation start site was cloned (Fig. 5B). When transfected into RK3E cell lines, a significantly higher level of reporter gene transcription was observed in the parental cell line than in RK3E-derived cell lines transformed by oncogenes (Fig. 5C). As no colon or pancreatic adenocarcinoma cell line with high endogenous DRO1 expression was available, the intestinal carcinoid cell line Colo320 (51) was used for analysis of regulation of DRO1 expression in human cancer as its DRO1 mRNA expression level corresponds to levels observed in normal colonic epithelium (data not shown). Transfection of the full-length DRO1 reporter (4235) and a construct lacking 3068 bases upstream of a KpnI site (1166) resulted in a significant increase of reporter gene activity in the colorectal cancer cell lines Caco2, HCT116, and SW480, but not in the Colo320 line (Fig. 5D). These data suggest that the low levels of DRO1 expression in cancer cell lines might at least in part be accountable to trans-acting repressors binding to the DRO1 promoter. To further narrow down this putative binding region, the first 1732 bases of the promoter construct were deleted. Both in SW480 as well as in HCT116 cells this only led to a modest increase in transcriptional activation (Fig. 5E). However, further deletion of 473 bases resulted in transcription levels corresponding to the construct lacking the 3068 bases (Fig. 5E). These experiments define a region of less than 500 bases within the DRO1 promoter that most likely contains one or more binding sites for so far undefined trans-acting repressors.

Ectopic Expression of DRO1 in Cancer Cells Lacking Endogenous DRO1 Expression Reduces Malignant Growth Properties—Two colorectal cancer cell lines, HCT116 and SW480, and two pancreatic cancer cell lines, BxPc3 and Panc I, with very minimal endogenous DRO1 expression were transfected with a plasmid encoding both DRO1 and neomycin resistance or a plasmid encoding neomycin resistance only. After transfection, cells were selected in G418 and tissue culture plates were monitored for appearance of colonies, cultures were stained and colonies were counted. DRO1 expression reduced G418-resistant colony formation by 62% in HCT116 cells and by 51% in SW480 cells (Fig. 6, A–D and J). In the pancreatic cancer cell lines Panc I and BxPc3, G418-resistant colony formation was reduced by 51 and 58% (Fig. 6, E–H and J), respectively. These data indicate that re-expression of DRO1 in cells with very minimal endogenous DRO1 expression can impair growth capabilities.



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  		FIG. 6.
Re-expression of DRO1 reduces clonogenic capacities of colorectal and pancreatic cancer cells. A–H, HCT116 and SW480 colorectal cancer cell lines and Panc I and BxPc3 pancreatic cancer cell lines were transfected with a DRO1 expression vector or control vector and selected for neomycin resistance. Three weeks after transfection plates were stained, counted, and photographed. J, quantitative analysis of colony formation in cancer cell lines. Relative colony formation compared with control plates was determined. Error bars represent mean ± S.E. of a triplicate experiment.

 
One {beta}-catenin and one K-Ras transformed RK3E line and the human cancer cell lines HCT116 and BxPc3 were chosen for further studies of the growth inhibitory effects of DRO1 re-expression. Clones with stable ectopic expression of DRO1 and clonal drug-resistant control lines were generated. Expression of the hemagglutinin-tagged DRO1 protein was documented by Western blotting (Fig. 7A). To further characterize the growth inhibitory effects of DRO1 in cancer cells, growth rates of the clonal lines expressing DRO1 were determined and compared with those of the control lines. Under standard cell culture conditions no significant differences in proliferation rates or cell cycle distribution were noted (data not shown). Next, the effect of DRO1 on anchorage-independent growth was studied. RK3E-{beta}-catenin/S33Y-A, RK3E-K-Ras, HCT116, and BxPc3 drug-resistant control clones and the corresponding parental cell lines readily formed colonies in agar (Fig. 7B, a, b, e, f, i, k, n, and o, and data not shown). In contrast, clones expressing DRO1 showed markedly reduced growth in agar (Fig. 7B, c, d, g, h, l, m, p, and q). Overall, DRO1 re-expression in oncogenic transformed RK3E as well as in HCT116 and BxPc3 cancer cell lines resulted in a reduction of colony formation in agar by more than 80% when compared with control cells (Fig. 7C). In consequence, as re-expression of DRO1 did not compromise cellular growth under standard adherent conditions but inhibited anchorage independent growth in multiple independent cell lines, DRO1 must be assumed to exert a negative selective pressure on non-adherent cells.



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  		FIG. 7.
DRO1 mediates growth suppression in cancer cells. A, clonal cell lines stably expressing DRO1 and drug-resistant control cell lines (designated with the suffices Neo and Puro for the resistance genes used, respectively) were established in HCT116, BxPc3, and RK3E cells transformed by S33Y-{beta}-catenin or K-Ras. Expression of hemagglutinin-tagged DRO1 was verified by Western blot and is shown in the upper panels of lanes 1–4 for HCT116, lanes 5–8 for BxPc3, lanes 9–12 for RK3E {beta}-catenin, and lanes 13–16 for RK3E-K-Ras. The blots were stripped, and ECL Western blot analysis with an anti-{beta}-actin antibody was carried out to demonstrate equal loading of the lanes (lower panel). B, the clonal cell lines were plated in soft agar and monitored for colony formation for 3 weeks. Then, plates were stained, counted and photographed. Control clones HCT116/Neo 1 and 2 (a and b), BxPc3/Neo 5 and 6 (e and f), RK3E-{beta}-catenin/Puro 9 and 10 (j and k) as well as RK3E-K-Ras/Puro 13 and 14 (n and o) readily formed macroscopically visible colonies. The DRO1 expressing clones HCT116/DRO1 3 and 4 (c and d), BxPc3/DRO1 7 and 8 (g and h), RK3E-{beta}-catenin/DRO1 11 and 12 (l and m) as well as RK3E-K-Ras/DRO1 15 and 16 (p and q) formed few, mostly only microscopically visible colonies. C, mean ± S.E. of colony numbers obtained in triplicate experiments for HCT116, BxPc3, RK3E {beta}-catenin, and RK3E K-Ras clonal lines are shown.

 
Sensitization of DRO1 Expressing Cells to Apoptosis—To study the mechanism underlying DRO1 mediated inhibition of colony formation in agar, cells were maintained in suspension mimicking anchorage independent growth conditions. Compared with standard adherent growth conditions, this did not result in significant differences in the cell cycle distribution between different clonal cell lines (data not shown). However, growth in suspension led to a much more pronounced increase of sub-G1 events in cell lines that express DRO1 compared with control cell lines (Fig. 8A and data not shown). The increased level of apoptosis in DRO1-expressing cells in suspension was confirmed by determination of caspase 3 activity in comparison to control cells (Fig. 8B). To test whether DRO1 expressing cells also show a higher susceptibility to other apoptotic stimuli when growing attached to the cell culture dish, cells were incubated with agonistic anti-APO-1/CD95 antibody. Whereas control cells were only moderately responsive to this apoptotic stimulus, apoptosis rates in DRO1-expressing cells were stimulated dose dependently to 55–60% (Fig. 8C and data not shown). Increased cleavage of caspase 8 and the downstream executioner caspase 3 was documented by Western blotting (Fig. 8D). In summary, these data indicate that DRO1 does not directly inhibit cellular growth or induce apoptosis but sensitizes cells to apoptotic signals.



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  		FIG. 8.
Re-expression of DRO1 sensitizes cells to anoikis and CD95-induced apoptosis. A, cells were cultured in suspension for 24 h. Then nuclei were stained with propidium iodide and fluorescence-activated cell sorter analysis was performed. Mean values ± S.E. of sub-G1 events of a triplicate experiment relative to total cell counts are shown. B, determination of caspase-3 activity in cells after 24 h in suspension. Mean ± S.E. of a triplicate experiment are shown. C, adherent cells were treated with the indicated concentrations of agonistic anti-APO1/CD95 antibody for 24 h. Then analysis of sub-G1 events by fluorescence-activated cell sorter analysis was performed. Mean ± S.E. of a triplicate experiment are shown. D, cleavage of caspases 3 and 8 in cells treated with 500 ng/ml agonistic anti-APO1/CD95 antibody is demonstrated by ECL Western blot analysis. After incubation with the caspase 8 antibody (upper panel) the membrane was stripped twice and successively incubated with the antibodies against caspase 3 (middle panel) and {beta}-actin as a loading control (lower panel). The cleavage products of the caspases are indicated by arrows.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Transformation of normal epithelial cells by oncogenes results in fundamental changes in the transcriptional program of a cell. The Wnt/APC/{beta}-catenin/TCF pathway is frequently deregulated in human cancers, with more than 80% of colorectal cancers showing constitutive activation (for review see Ref. 52). Transcriptional changes resulting from {beta}-catenin stabilization and constitutive formation of {beta}-catenin·TCF complexes have been intensively studied (for review see Ref. 53). Expression of most of the so far identified {beta}-catenin/TCF target genes is elevated as a result of {beta}-catenin stabilization. Only a few studies have focused on the identification of genes negatively regulated during colorectal carcinogenesis in general and in the Wnt/APC/{beta}-catenin/TCF pathway in particular. The DRCTNNB1A (27), MCP-3 (28), and IGFBP-6 (29) genes have been identified as genes whose expression may be negatively regulated by {beta}-catenin/TCF. However, as demonstrated in a recently published large microarray based analysis of the Wnt/APC/{beta}-catenin/TCF pathway, the transcriptional response to {beta}-catenin stabilization involves up-regulation and down-regulation of numerous genes (54). On the one hand, several of these down-regulated genes represented differentiation markers such as alkaline phosphatase and mucin-2 (54). On the other hand, expression of the cell cycle inhibitor p21Cip1 was found to be strongly suppressed as a result of {beta}-catenin deregulation. Although this is an indirect effect of the Wnt/APC/{beta}-catenin/TCF pathway and several pathways converge in the regulation of p21, evidence has been presented demonstrating a pivotal role of p21 down-regulation in colorectal carcinogenesis (54).

Here, we describe the identification of DRO1, a gene that is down-regulated in {beta}-catenin-transformed RK3E cells. Down-regulation of DRO1 was not found to be a unique effect of {beta}-catenin, as RK3E cells transformed by other oncogenes including RAS proteins, GLI, and c-MYC also revealed reduced expression levels of DRO1. DRO1 expression was found to be substantially reduced in the majority of colon and pancreatic cancer cells as well as in primary colorectal cancers compared with adjacent normal epithelium. Our studies yielded evidence that down-regulation of DRO1 in transformed RK3E lines and human cancer cell lines was not likely to be a direct effect of the oncogenes. A putative CpG island in the DRO1 gene (not meeting high stringency criteria) was found to be methylated in both normal colonic epithelium and colorectal cancer cell lines. Treatment of cells lacking endogenous DRO1 expression with the demethylating agent azacytidine resulted in a minor increase in DRO1 expression only. Therefore, direct regulation of DRO1 expression by DNA hypermethylation is also unlikely. In addition, DRO1 reporter gene assays suggest that down-regulation of DRO1 in human cancer is at least in part because of transcriptional repression.

DRO1 sequence analysis revealed that rat and human DRO1 might be the orthologues of the mouse Urb and the chick equarin genes. Urb was identified as being up-regulated in the brown adipose tissue of a mouse model of metabolic syndrome, i.e. bombesin receptor subtype-3-deficient mice, and was therefore named Urb for "up-regulated in bombesin receptor subtype-3 deficient mice" (40). Almost ubiquitous expression was demonstrated and Urb was hypothesized to regulate thermogenesis in brown adipose tissue and food intake as a secreted mediator. Recently, a chick gene was cloned in a screen for soluble proteins in the embryonal eye lens. Because of its expression along the equatorial region of the lens it was named Equarin (41). Primary amino acid sequence analysis revealed high similarity to mouse Urb and rat as well as human DRO1. Two different splice variants were identified, one corresponding to full-length DRO1 (Equarin-L) and an alternatively spliced carboxyl terminal-truncated form (Equarin-S). No similar alternatively spliced variants that would lead to carboxyl-terminal-truncated forms of DRO1 are present in the databases of rat or human expressed sequence tag databases to date. Most interestingly, equarin expression was found to be asymmetric within the embryonic lens with a strong dorso-ventral gradient suggesting a morphogenetic function. When injected ectopically in Xenopus embryos equarin was able to disturb normal retinal development and prevented closure of the optical fissure. In contrast to the chick orthologue equarin and the presence of a presumptive signal peptide we did not find DRO1 to be secreted in significant amounts. Immunofluorescence studies revealed cytosolic localization and only partial overlap with markers for the endoplasmic reticulum and Golgi stack was seen. However, post-translational modification by N-glycosylation could be demonstrated suggesting that DRO1 is at least partly processed in the endoplasmic reticulum.

Although a high degree of identity (>97%) between rat Dro1 and transcripts of the gene Ssg1 are present throughout the sequence, subtle differences in comparison to the now available rat genomic sequences and DRO1 sequence led Marcantonio et al. (55, 56) to suggest that the SSG1 protein has only 385 amino acids, which are identical to the central portion of rat DRO1 (aa 403–751 of rat DRO1).

The DRO1 open reading frame of human and rat contains three repeats with significant homology to the carboxyl-terminal region of the putative tumor suppressor gene DRS/SRPX/ETX1 and the gene SRPUL. Human DRS/SRPX/ETX1 sequence was temporarily supposed and later excluded as a candidate gene for X-linked retinitis pigmentosa. It was therefore first referred to as "sushi repeat protein on the X-chromosome" (SRPX (43, 57) and ETX1 (42)). Later transformation studies with various oncogenes using a rat fibroblastic cell line led to the identification of rat Drs ("down-regulated by v-src") (44). Drs has subsequently been shown to inhibit the transformation of a fibroblastic cell line by c-Src and to reduce anchorage independent growth of cancer cells in soft agar (58, 59). Deletion constructs of Drs mapped the essential region for its function to the domain that is homologous to the three repeats of DRO1. Each of the three internal repeats of DRO1 is about 30% identical to the carboxyl-terminal region of Drs, which has been suggested to contain a transmembrane region. However, no convincing candidate transmembrane region of DRO1 could be identified by computerized algorithms (60). Given the fact that the three internal repeats of DRO1 are by far the most conserved regions of the gene between man and rat, it is likely that the sequence similarity with the carboxyl terminus of Drs represents the conservation of a functionally important region. The three repeats of DRO1 also show similarity to a close relative of Drs, which has been cloned as a target gene of the promyelocytic leukemia fusion protein E2A-HLF and was therefore named SRPUL ("sushi repeat protein up-regulated in leukemia") (45). Comparison of the three repeats of human, rat, and putative zebrafish DRO1 (GenBankTM accession number CAC94894 [GenBank] , mouse Urb, and chick Equarin-L with the sequences of rat, human, and mouse Drs/SRPX/ETX 1 as well as human SRPUL, revealed a striking conservation of 13 amino acid residues. We therefore suggest that DRO1, URB, equarin, DRS/SRPX/ETX1, and SRPUL should be considered members of a new protein family with a DUDES (DRO1-URB-DRS-equarin-SRPUL) repeat.

Upon re-expression of DRO1 in cancer cell lines lacking relevant endogenous DRO1 expression, a reduction of malignant growth properties was observed: colony formation and anchorage independent growth were significantly compromised in a variety of cell lines. However, no negative influence of DRO1 re-expression on proliferation, cell cycle distribution, or apoptosis was noted under normal adherent culture conditions. In contrast, DRO1 was found to specifically inhibit anchorage independent growth by induction of detachment-induced apoptosis, a phenomenon also known as anoikis (reviewed in Ref. 61). Cells overexpressing DRO1 were also more prone to undergo apoptosis when stimulated with agonistic anti-APO-1/CD95 antibody suggesting sensitization to different apoptotic stimuli. This may be of importance for tissue homeostasis as well carcinogenesis. Intestinal epithelial cells are derived from stem cells at the bottom of the crypts. They undergo apoptosis after a total lifetime of 3–5 days once they reach the luminal surface and loose their anchorage to the extracellular matrix (62). Apoptosis upon detachment from the extracellular matrix is therefore essential for the maintenance of the normal architecture of the intestine. Therefore, the acquisition of resistance to loss of cell anchorage and anoikis is regarded as a critical step during neoplastic transformation (63). In addition, it has been recognized that one of the hallmarks of cancer cells is the resistance to apoptotic stimuli as diverse as detachment from their normal extracellular matrix, withdrawal of growth factors, or stimulation of death receptors (64).

Similar to DRO1, expression of Drs is also down-regulated in various human cancers and re-expression of Drs in cancer cells lacking endogenous Drs expression results in suppression of anchorage independent growth (46, 47, 65). As DRO1 expression is down-regulated in human cancer and re-expression of DRO1 makes cancer cells susceptible to apoptotic stimuli, DRO1 can be viewed as a candidate tumor suppressor gene.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY548105 [GenBank] , AY548106 [GenBank] , AY548107 [GenBank] .

* This work was supported by Deutsche Forschungsgemeinschaft Grants KO 1826/2-1 and KO 1826/2-3 and a FöFoLe grant from the Faculty of Medicine of the University of Munich. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Present address: Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, MI 48105. Back

§§ To whom correspondence should be addressed: Dept. of Medicine II, Klinikum Grosshadern, University of Munich, Marchioninistrasse 15, 81377 Munich, Germany. E-mail: fkolligs{at}med.uni-muenchen.de.

1 The abbreviations used are: APC, adenomatous polyposis coli; TCF, T-cell factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; bGH, bovine growth hormone; RT, reverse transcriptase; aa, amino acid(s); APP, {beta}-amyloid precursor protein; DRO1, down-regulated by oncogenes 1. Back

2 Julenius, K., Molgaard, A., Gupta, R., and Brunak, S. (2005) Glycobiology 15, 153–164. Back


   ACKNOWLEDGMENTS
 
We thank G. van Kaick (DKFZ, Heidelberg), H. Lahm (Thoraxklinik Heidelberg, Heidelberg), R. Ehehalt (University of Heidelberg, Heidelberg), Peter H. Krammer (Deutsches Krebsforschungszentrum, Heidelberg), M. Erhard (University of Munich, Munich), and E. Wolf (University of Munich, Munich) for sharing reagents with us. We appreciated the help of A. Vollmar and W. Roedl (University of Munich, Munich) with confocal microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fearon, E. R., Hamilton, S. R., and Vogelstein, B. (1987) Science 238, 193-197[Abstract/Free Full Text]
  2. Jones, P. A. (2003) Ann. N. Y. Acad. Sci. 983, 213-219[CrossRef][Medline] [Order article via Infotrieve]
  3. Bardeesy, N., and DePinho, R. A. (2002) Nat. Rev. Cancer 2, 897-909[CrossRef][Medline] [Order article via Infotrieve]
  4. Fearon, E. R., and Vogelstein, B. (1990) Cell 61, 759-767[CrossRef][Medline] [Order article via Infotrieve]
  5. Bienz, M., and Clevers, H. (2000) Cell 103, 311-320[CrossRef][Medline] [Order article via Infotrieve]
  6. Kolligs, F. T., Bommer, G., and Goke, B. (2002) Digestion 66, 131-144[CrossRef][Medline] [Order article via Infotrieve]
  7. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., Hughes, J. P., Warrington, J., McPherson, J., Wasmuth, J., LePashier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. (1991) Cell 66, 589-600[CrossRef][Medline] [Order article via Infotrieve]
  8. Kinzler, K. W., Nilbert, M. C., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. C., Hamilton, S. R., Hedge, P., Markham, A., Carlson, M., Joslyn, G., Groden, J., White, R., Mini, Y., Miyoshi, Y., Nishisho, I., and Nakamura, Y. (1991) Science 251, 1366-1370[Abstract/Free Full Text]
  9. Nishisho, I., Nakamura, Y., Miyoshi, Y., Miki, Y., Ando, H., Horii, A., Koyama, K., Utsunomiya, J., Baba, S., and Hedge, P. (1991) Science 253, 665-669[Abstract/Free Full Text]
  10. Morin, P. J., Sparks, A. B., Korinek, V., Barker, N., Clevers, H., Vogelstein, B., and Kinzler, K. W. (1997) Science 275, 1787-1790[Abstract/Free Full Text]
  11. Ilyas, M., Tomlinson, I. P., Rowan, A., Pignatelli, M., and Bodmer, W. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10330-10334[Abstract/Free Full Text]
  12. Koch, A., Denkhaus, D., Albrecht, S., Leuschner, I., von Schweinitz, D., and Pietsch, T. (1999) Cancer Res. 59, 269-273[Abstract/Free Full Text]
  13. Wei, Y., Fabre, M., Branchereau, S., Gauthier, F., Perilongo, G., and Buendia, M. A. (2000) Oncogene 19, 498-504[CrossRef][Medline] [Order article via Infotrieve]
  14. de La Coste, A., Romagnolo, B., Billuart, P., Renard, C. A., Buendia, M. A., Soubrane, O., Fabre, M., Chelly, J., Beldjord, C., Kahn, A., and Perret, C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8847-8851[Abstract/Free Full Text]
  15. Miyoshi, Y., Iwao, K., Nagasawa, Y., Aihara, T., Sasaki, Y., Imaoka, S., Murata, M., Shimano, T., and Nakamura, Y. (1998) Cancer Res. 58, 2524-2527[Abstract/Free Full Text]
  16. Park, W. S., Oh, R. R., Park, J. Y., Lee, S. H., Shin, M. S., Kim, Y. S., Kim, S. Y., Lee, H. K., Kim, P. J., Oh, S. T., Yoo, N. J., and Lee, J. Y. (1999) Cancer Res. 59, 4257-4260[Abstract/Free Full Text]
  17. Abraham, S. C., Wu, T. T., Hruban, R. H., Lee, J. H., Yeo, C. J., Conlon, K., Brennan, M., Cameron, J. L., and Klimstra, D. S. (2002) Am. J. Pathol. 160, 953-962[Abstract/Free Full Text]
  18. Abraham, S. C., Klimstra, D. S., Wilentz, R. E., Yeo, C. J., Conlon, K., Brennan, M., Cameron, J. L., Wu, T. T., and Hruban, R. H. (2002) Am. J. Pathol. 160, 1361-1369[Abstract/Free Full Text]
  19. He, T. C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998) Science 281, 1509-1512[Abstract/Free Full Text]
  20. Tetsu, O., and McCormick, F. (1999) Nature 398, 422-426[CrossRef][Medline] [Order article via Infotrieve]
  21. Shtutman, M., Zhurinsky, J., Simcha, I., Albanese, C., D'Amico, M., Pestell, R., and Ben-Ze'ev, A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5522-5527[Abstract/Free Full Text]
  22. Kolligs, F. T., Nieman, M. T., Winer, I., Hu, G., Van Mater, D., Feng, Y., Smith, I. M., Wu, R., Zhai, Y., Cho, K. R., and Fearon, E. R. (2002) Cancer Cell 1, 145-155[CrossRef][Medline] [Order article via Infotrieve]
  23. van der Heyden, M. A., Rook, M. B., Hermans, M. M., Rijksen, G., Boonstra, J., Defize, L. H., and Destree, O. H. (1998) J. Cell Sci. 111, 1741-1749[Abstract]
  24. Leung, J. Y., Kolligs, F. T., Wu, R., Zhai, Y., Kuick, R., Hanash, S., Cho, K. R., and Fearon, E. R. (2002) J. Biol. Chem. 277, 21657-21665[Abstract/Free Full Text]
  25. Jho, E. H., Zhang, T., Domon, C., Joo, C. K., Freund, J. N., and Costantini, F. (2002) Mol. Cell. Biol. 22, 1172-1183[Abstract/Free Full Text]
  26. Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van de Wetering, M., Clevers, H., Schlag, P. M., Birchmeier, W., and Behrens, J. (2002) Mol. Cell. Biol. 22, 1184-1193[Abstract/Free Full Text]
  27. Kawasoe, T., Furukawa, Y., Daigo, Y., Nishiwaki, T., Ishiguro, H., Fujita, M., Satoh, S., Miwa, N., Nagasawa, Y., Miyoshi, Y., Ogawa, M., and Nakamura, Y. (2000) Cancer Res. 60, 3354-3358[Abstract/Free Full Text]
  28. Fujita, M., Furukawa, Y., Nagasawa, Y., Ogawa, M., and Nakamura, Y. (2000) Cancer Res. 60, 6683-6687[Abstract/Free Full Text]
  29. Denys, H., Jadidizadeh, A., Amini Nik, S., Van Dam, K., Aerts, S., Alman, B. A., Cassiman, J. J., and Tejpar, S. (2004) Oncogene 23, 654-664[CrossRef][Medline] [Order article via Infotrieve]
  30. Ruppert, J. M., Vogelstein, B., and Kinzler, K. W. (1991) Mol. Cell. Biol. 11, 1724-1728[Abstract/Free Full Text]
  31. Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., and Siebert, P. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6025-6030[Abstract/Free Full Text]
  32. Kolligs, F. T., Hu, G., Dang, C. V., and Fearon, E. R. (1999) Mol. Cell. Biol. 19, 5696-5706[Abstract/Free Full Text]
  33. Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract/Free Full Text]
  34. Kolligs, F. T., Kolligs, B., Hajra, K. M., Hu, G., Tani, M., Cho, K. R., and Fearon, E. R. (2000) Genes Dev. 14, 1319-1331[Abstract/Free Full Text]
  35. Saha, S., Bardelli, A., Buckhaults, P., Velculescu, V. E., Rago, C., St. Croix, B., Romans, K. E., Choti, M. A., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (2001) Science 294, 1343-1346[Abstract/Free Full Text]
  36. Muller, P. Y., Janovjak, H., Miserez, A. R., and Dobbie, Z. (2002) BioTechniques 32, 1372-1379[Medline] [Order article via Infotrieve]
  37. Pfaffl, M. W. (2001) Nucleic Acids Res. 29, e45[Abstract/Free Full Text]
  38. Ehehalt, R., Michel, B., De Pietri Tonelli, D., Zacchetti, D., Simons, K., and Keller, P. (2002) Biochem. Biophys. Res. Commun. 293, 30-37[CrossRef][Medline] [Order article via Infotrieve]
  39. Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. (1991) J. Immunol. Methods 139, 271-279[CrossRef][Medline] [Order article via Infotrieve]
  40. Aoki, K., Sun, Y. J., Aoki, S., Wada, K., and Wada, E. (2002) Biochem. Biophys. Res. Commun. 290, 1282-1288[CrossRef][Medline] [Order article via Infotrieve]
  41. Mu, H., Ohta, K., Kuriyama, S., Shimada, N., Tanihara, H., Yasuda, K., and Tanaka, H. (2003) Mech. Dev. 120, 143-155[CrossRef][Medline] [Order article via Infotrieve]
  42. Dry, K. L., Aldred, M. A., Edgar, A. J., Brown, J., Manson, F. D., Ho, M. F., Prosser, J., Hardwick, L. J., Lennon, A. A., and Thomson, K. (1995) Hum. Mol. Genet. 4, 2347-2353[Abstract/Free Full Text]
  43. Meindl, A., Carvalho, M. R., Herrmann, K., Lorenz, B., Achatz, H., Lorenz, B., Apfelstedt-Sylla, E., Wittwer, B., Ross, M., and Meitinger, T. (1995) Hum. Mol. Genet. 4, 2339-2346[Abstract/Free Full Text]
  44. Pan, J., Nakanishi, K., Yutsudo, M., Inoue, H., Li, Q., Oka, K., Yoshioka, N., and Hakura, A. (1996) FEBS Lett. 383, 21-25[CrossRef][Medline] [Order article via Infotrieve]
  45. Kurosawa, H., Goi, K., Inukai, T., Inaba, T., Chang, K. S., Shinjyo, T., Rakestraw, K. M., Naeve, C. W., and Look, A. T. (1999) Blood 93, 321-332[Abstract/Free Full Text]
  46. Shimakage, M., Takami, K., Kodama, K., Mano, M., Yutsudo, M., and Inoue, H. (2002) Hum. Pathol. 33, 615-619[CrossRef][Medline] [Order article via Infotrieve]
  47. Shimakage, M., Kawahara, K., Kikkawa, N., Sasagawa, T., Yutsudo, M., and Inoue, H. (2000) Int. J. Cancer 87, 5-11[CrossRef][Medline] [Order article via Infotrieve]
  48. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Protein Eng. 10, 1-6[Abstract/Free Full Text]
  49. Sigrist, C. J., Cerutti, L., Hulo, N., Gattiker, A., Falquet, L., Pagni, M., Bairoch, A., and Bucher, P. (2002) Brief Bioinform. 3, 265-274[Abstract/Free Full Text]
  50. Wang, Y., and Leung, F. C. (2004) Bioinformatics 20, 1170-1177[Abstract/Free Full Text]
  51. Quinn, L. A., Moore, G. E., Morgan, R. T., and Woods, L. K. (1979) Cancer Res. 39, 4914-4924[Abstract/Free Full Text]
  52. Polakis, P. (2000) Genes Dev. 14, 1837-1851[Free Full Text]
  53. Giles, R. H., van Es, J. H., and Clevers, H. (2003) Biochim. Biophys. Acta 1653, 1-24[Medline] [Order article via Infotrieve]
  54. van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A. P., Tjon-Pon-Fong, M., Moerer, P., van den Born, M., Soete, G., Pals, S., Eilers, M., Medema, R., and Clevers, H. (2002) Cell 111, 241-250[CrossRef][Medline] [Order article via Infotrieve]
  55. Marcantonio, D., Chalifour, L. E., Alaoui-Jamali, M. A., and Huynh, H. T. (2001) J. Mol. Endocrinol. 26, 175-184[Abstract]
  56. Marcantonio, D., Chalifour, L. E., Alaoui-Jamali, M. A., Alpert, L., and Huynh, H. T. (2001) Endocrinology 142, 2409-2418[Abstract/Free Full Text]
  57. El Nemer, W., Colin, Y., Collec, E., Gane, P., Cartron, J. P., and Kim, C. L. (2000) Eur. J. Immunogenet. 27, 29-33[CrossRef][Medline] [Order article via Infotrieve]
  58. Inoue, H., Pan, J., and Hakura, A. (1998) J. Virol. 72, 2532-2537[Abstract/Free Full Text]
  59. Yamashita, A., Hakura, A., and Inoue, H. (1999) Oncogene 18, 4777-4787[CrossRef][Medline] [Order article via Infotrieve]
  60. Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. (2001) J. Mol. Biol. 305, 567-580[CrossRef][Medline] [Order article via Infotrieve]
  61. Grossmann, J. (2002) Apoptosis 7, 247-260[CrossRef][Medline] [Order article via Infotrieve]
  62. Grossmann, J., Walther, K., Artinger, M., Rummele, P., Woenckhaus, M., and Scholmerich, J. (2002) Am. J. Gastroenterol. 97, 1421-1428[CrossRef][Medline] [Order article via Infotrieve]
  63. Ruoslahti, E., and Reed, J. C. (1994) Cell 77, 477-478[CrossRef][Medline] [Order article via Infotrieve]
  64. Hanahan, D., and Weinberg, R. A. (2000) Cell 100, 57-70[CrossRef][Medline] [Order article via Infotrieve]
  65. Kim, C. J., Shimakage, M., Kushima, R., Mukaisho, K., Shinka, T., Okada, Y., and Inoue, H. (2003) Hum. Pathol. 34, 654-657[CrossRef][Medline] [Order article via Infotrieve]

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